BGC Atlas: a web resource for exploring the global chemical diversity encoded in bacterial genomes.

Secondary metabolites are compounds not essential for an organism's development, but provide significant ecological and physiological benefits. These compounds have applications in medicine, biotechnology and agriculture. Their production is encoded in biosynthetic gene clusters (BGCs), groups of genes collectively directing their biosynthesis. The advent of metagenomics has allowed researchers to study BGCs directly from environmental samples, identifying numerous previously unknown BGCs encoding unprecedented chemistry. Here, we present the BGC Atlas (https://bgc-atlas.cs.uni-tuebingen.de), a web resource that facilitates the exploration and analysis of BGC diversity in metagenomes. The BGC Atlas identifies and clusters BGCs from publicly available datasets, offering a centralized database and a web interface for metadata-aware exploration of BGCs and gene cluster families (GCFs). We analyzed over 35 000 datasets from MGnify, identifying nearly 1.8 million BGCs, which were clustered into GCFs. The analysis showed that ribosomally synthesized and post-translationally modified peptides are the most abundant compound class, with most GCFs exhibiting high environmental specificity. We believe that our tool will enable researchers to easily explore and analyze the BGC diversity in environmental samples, significantly enhancing our understanding of bacterial secondary metabolites, and promote the identification of ecological and evolutionary factors shaping the biosynthetic potential of microbial communities.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes.

Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. KEY POINTS: • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.

Decoding the genome of superior chapatti quality Indian wheat variety 'C 306' unravelled novel genomic variants for chapatti and nutrition quality related genes.

An Indian wheat variety, 'C 306' has good chapatti quality, which is controlled by multiple genes that have not been explored. We report the high quality de novo assembled genome of 'C 306' by combining short and long read sequencing data. The hybrid assembly covered 93% of gene space and identified about 142 K coding genes, 34% repetitive DNA and ~ 501 K SSR motifs. The phylogenetic analysis of about 83 K orthologous protein groups suggested the closest relationship with T. turgidum, T. aestivum and Ae. tauschii. Genome wide analysis annotated 69,217,536 genomic variants. Out of them, 1423 missense and 117 deleterious variants identified in processing, nutrition, and chapatti quality related genes such as alpha- and beta-gliadin, SSI, SSIII, SUT1, SBEI, CHS, YSL, DMAS, and NAS encoded proteins. These variants may affect quality genes. The genomic data will be potential genomic resources in wheat breeding programs for quality improvement.

Physiological and molecular responses to combinatorial iron and phosphate deficiencies in hexaploid wheat seedlings.

Iron (Fe) and phosphorus (P) are the essential mineral nutrients for plant growth and development. However, the molecular interaction of the Fe and P pathways in crops remained largely obscure. In this study, we provide a comprehensive physiological and molecular analysis of hexaploid wheat response to single (Fe, P) and its combinatorial deficiencies. Our data showed that inhibition of the primary root growth occurs in response to Fe deficiency; however, growth was rescued when combinatorial deficiencies occurred. Analysis of RNAseq revealed that distinct molecular rearrangements during combined deficiencies with predominance for genes related to metabolic pathways and secondary metabolite biosynthesis primarily include genes for UDP-glycosyltransferase, cytochrome-P450s, and glutathione metabolism. Interestingly, the Fe-responsive cis-regulatory elements in the roots in Fe stress conditions were enriched compared to the combined stress. Our metabolome data also revealed the accumulation of distinct metabolites such as amino-isobutyric acid, arabinonic acid, and aconitic acid in the combined stress environment. Overall, these results are essential in developing new strategies to improve the resilience of crops in limited nutrients.

The confluence of big data and evolutionary genome mining for the discovery of natural products.

This review covers literature between 2003-2021The development and application of genome mining tools has given rise to ever-growing genetic and chemical databases and propelled natural products research into the modern age of Big Data. Likewise, an explosion of evolutionary studies has unveiled genetic patterns of natural products biosynthesis and function that support Darwin's theory of natural selection and other theories of adaptation and diversification. In this review, we aim to highlight how Big Data and evolutionary thinking converge in the study of natural products, and how this has led to an emerging sub-discipline of evolutionary genome mining of natural products. First, we outline general principles to best utilize Big Data in natural products research, addressing key considerations needed to provide evolutionary context. We then highlight successful examples where Big Data and evolutionary analyses have been combined to provide bioinformatic resources and tools for the discovery of novel natural products and their biosynthetic enzymes. Rather than an exhaustive list of evolution-driven discoveries, we highlight examples where Big Data and evolutionary thinking have been embraced for the evolutionary genome mining of natural products. After reviewing the nascent history of this sub-discipline, we discuss the challenges and opportunities of genomic and metabolomic tools with evolutionary foundations and/or implications and provide a future outlook for this emerging and exciting field of natural product research.

Integrative analysis of hexaploid wheat roots identifies signature components during iron starvation.

Iron (Fe) is an essential micronutrient for all organisms. In crop plants, Fe deficiency can decrease crop yield significantly; however, our current understanding of how major crops respond to Fe deficiency remains limited. Herein, the effect of Fe deprivation at both the transcriptomic and metabolic level in hexaploid wheat was investigated. Genome-wide gene expression reprogramming was observed in wheat roots subjected to Fe starvation, with a total of 5854 genes differentially expressed. Homoeologue and subgenome-specific analysis unveiled the induction-biased contribution from the A and B genomes. In general, the predominance of genes coding for nicotianamine synthase, yellow stripe-like transporters, metal transporters, ABC transporters, and zinc-induced facilitator-like protein was noted. Expression of genes related to the Strategy II mode of Fe uptake was also predominant. Our transcriptomic data were in agreement with the GC-MS analysis that showed the enhanced accumulation of various metabolites such as fumarate, malonate, succinate, and xylofuranose, which could be contributing to Fe mobilization. Interestingly, Fe starvation leads to a significant temporal increase of glutathione S-transferase at both the transcriptional level and enzymatic activity level, which indicates the involvement of glutathione in response to Fe stress in wheat roots. Taken together, our result provides new insight into the wheat response to Fe starvation at the molecular level and lays the foundation to design new strategies for the improvement of Fe nutrition in crops.

Isomalto-oligosaccharides, a prebiotic, functionally augment green tea effects against high fat diet-induced metabolic alterations via preventing gut dysbacteriosis in mice.

High fat diet (HFD)-induced alterations in gut microbiota and resultant 'leaky gut' phenomenon promotes metabolic endotoxemia, ectopic fat deposition, and low-grade systemic inflammation. Here we evaluated the effects of a combination of green tea extract (GTE) with isomalto-oligosaccharide (IMOs) on HFD-induced alterations in mice. Male Swiss albino mice were fed with HFD (58% fat kcal) for 12 weeks. Systemic adiposity, gut derangement parameters and V3-V4 region based 16S rRNA metagenomic sequencing, ectopic fat deposition, liver metabolome analysis, systemic and tissue inflammation, and energy homeostasis markers along with gene expression analysis in multiple tissues were done in mice supplemented with GTE, IMOs or their combination. The combination of GTE and IMOs effectively prevented HFD-induced adiposity and lipid accumulation in liver and muscle while normalizing fasting blood glucose, insulin, glucagon, and leptin levels. Co-administration of GTE with IMOs effectively modulated liver metabolome associated with lipid metabolism. It also prevented leaky gut phenotype and HFD-induced increase in circulating lipopolysaccharides and pro-inflammatory cytokines (e.g. resistin, TNF-α, and IL-1ß) and reduction in anti-inflammatory cytokines (e.g. adiponectin and IL-6). Gene expression analysis across multiple tissues further supported these functional outcomes. Most importantly, this combination improved beneficial gut microbiota (Lactobacillus sp., Bifidobacteria, Akkermansia muciniphila, Roseburia spp.) abundances, restored Firmicutes/Bacteriodetes and improved Prevotella/Bacteroides proportions. In particular, a combination of these two agents has shown improved beneficial effects on multiple parameters studied. Data presented herein suggests that strategically chosen food components might be highly effective in the prevention of HFD-induced alterations and may further be developed as functional foods.

De novo assembly and characterization of transcriptomes of early-stage fruit from two genotypes of Annona squamosa L. with contrast in seed number.

BACKGROUND: Annona squamosa L., a popular fruit tree, is the most widely cultivated species of the genus Annona. The lack of transcriptomic and genomic information limits the scope of genome investigations in this important shrub. It bears aggregate fruits with numerous seeds. A few rare accessions with very few seeds have been reported for Annona. A massive pyrosequencing (Roche, 454 GS FLX+) of transcriptome from early stages of fruit development (0, 4, 8 and 12 days after pollination) was performed to produce expression datasets in two genotypes, Sitaphal and NMK-1, that show a contrast in the number of seeds set in fruits. The data reported here is the first source of genome-wide differential transcriptome sequence in two genotypes of A. squamosa, and identifies several candidate genes related to seed development. RESULTS: Approximately 1.9 million high-quality clean reads were obtained in the cDNA library from the developing fruits of both the genotypes, with an average length of about 568 bp. Quality-reads were assembled de novo into 2074 to 11004 contigs in the developing fruit samples at different stages of development. The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb. Assembled unigenes were functionally annotated by querying against the protein sequences of five different public databases (NCBI non redundant, Prunus persica, Vitis vinifera, Fragaria vesca, and Amborella trichopoda), with an E-value cut-off of 10(-5). A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database. These sequences could be genes specific to Annona sp. or belong to untranslated regions. Several of the unigenes representing pathways related to primary and secondary metabolism, and seed and fruit development expressed at a higher level in Sitaphal, the densely seeded cultivar in comparison to the poorly seeded NMK-1. A total of 2629 (Sitaphal) and 3445 (NMK-1) Simple Sequence Repeat (SSR) motifs were identified respectively in the two genotypes. These could be potential candidates for transcript based microsatellite analysis in A. squamosa. CONCLUSION: The present work provides early-stage fruit specific transcriptome sequence resource for A. squamosa. This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana.

BACKGROUND: The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level. RESULTS: In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5' regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes. CONCLUSIONS: This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.

Targeting MicroRNAs in Prevention and Treatment of Neurodegenerative Disorders.

Preclinical Research microRNAs (miRNAs) are small noncoding RNAs (ncRNAs) that are key regulators of gene expression. They act on wide range of targets by binding to mRNA via imperfect complementarity at 3' UTR. Evidence suggests that miRNAs regulate many biological processes including neuronal development, differentiation, and disease. Altered expression of several miRNAs has been reported in many neurodegenerative disorders (NDDs). Many miRNAs are altered in these diseases, but miRNA 15, miRNA 21, and miRNA 146a have been shown to play critical role in many neurodegenerative conditions. As these miRNAs regulate many genes, miRNA targeted approaches would allow concurrently targeting of multiple effectors of pathways that regulate disease progression. In this review, we describe the role of miRNAs in various NDDs and their potential as therapeutic tools in prevention and treatment of neurological conditions.

Genome-wide transcriptome study in wheat identified candidate genes related to processing quality, majority of them showing interaction (quality x development) and having temporal and spatial distributions.

BACKGROUND: The cultivated bread wheat (Triticum aestivum L.) possesses unique flour quality, which can be processed into many end-use food products such as bread, pasta, chapatti (unleavened flat bread), biscuit, etc. The present wheat varieties require improvement in processing quality to meet the increasing demand of better quality food products. However, processing quality is very complex and controlled by many genes, which have not been completely explored. To identify the candidate genes whose expressions changed due to variation in processing quality and interaction (quality x development), genome-wide transcriptome studies were performed in two sets of diverse Indian wheat varieties differing for chapatti quality. It is also important to understand the temporal and spatial distributions of their expressions for designing tissue and growth specific functional genomics experiments. RESULTS: Gene-specific two-way ANOVA analysis of expression of about 55 K transcripts in two diverse sets of Indian wheat varieties for chapatti quality at three seed developmental stages identified 236 differentially expressed probe sets (10-fold). Out of 236, 110 probe sets were identified for chapatti quality. Many processing quality related key genes such as glutenin and gliadins, puroindolines, grain softness protein, alpha and beta amylases, proteases, were identified, and many other candidate genes related to cellular and molecular functions were also identified. The ANOVA analysis revealed that the expression of 56 of 110 probe sets was involved in interaction (quality x development). Majority of the probe sets showed differential expression at early stage of seed development i.e. temporal expression. Meta-analysis revealed that the majority of the genes expressed in one or a few growth stages indicating spatial distribution of their expressions. The differential expressions of a few candidate genes such as pre-alpha/beta-gliadin and gamma gliadin were validated by RT-PCR. Therefore, this study identified several quality related key genes including many other genes, their interactions (quality x development) and temporal and spatial distributions. CONCLUSIONS: The candidate genes identified for processing quality and information on temporal and spatial distributions of their expressions would be useful for designing wheat improvement programs for processing quality either by changing their expression or development of single nucleotide polymorphisms (SNPs) markers.

Genome-wide Methylation Dynamics and Context-dependent Gene Expression Variability in Differentiating Preadipocytes.

Obesity, characterized by the accumulation of excess fat, is a complex condition resulting from the combination of genetic and epigenetic factors. Recent studies have found correspondence between DNA methylation and cell differentiation, suggesting a role of the former in cell fate determination. There is a lack of comprehensive understanding concerning the underpinnings of preadipocyte differentiation, specifically when cells are undergoing terminal differentiation (TD). To gain insight into dynamic genome-wide methylation, 3T3 L1 preadipocyte cells were differentiated by a hormone cocktail. The genomic DNA was isolated from undifferentiated cells and 4 hours, 2 days postdifferentiated cells, and 15 days TD cells. We employed whole-genome bisulfite sequencing (WGBS) to ascertain global genomic DNA methylation alterations at single base resolution as preadipocyte cells differentiate. The genome-wide distribution of DNA methylation showed similar overall patterns in pre-, post-, and terminally differentiated adipocytes, according to WGBS analysis. DNA methylation decreases at 4 hours after differentiation initiation, followed by methylation gain as cells approach TD. Studies revealed novel differentially methylated regions (DMRs) associated with adipogenesis. DMR analysis suggested that though DNA methylation is global, noticeable changes are observed at specific sites known as "hotspots." Hotspots are genomic regions rich in transcription factor (TF) binding sites and exhibit methylation-dependent TF binding. Subsequent analysis indicated hotspots as part of DMRs. The gene expression profile of key adipogenic genes in differentiating adipocytes is context-dependent, as we found a direct and inverse relationship between promoter DNA methylation and gene expression.

Genome-wide identification of microsatellites for mapping, genetic diversity and cross-transferability in wheat (Triticum spp).

Wheat (Triticum aestivum L.) is a crucial global staple crop, and is consistently being improved to enhance yield, disease resistance, and quality traits. However, the development of molecular markers is a challenging task due to its hexaploid genome. Molecular marker system such as simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) are helpful for breeding, but SNP has limitations due to its development cost and its conversion to breeder markers. The study proposed an in-silico approach, by utilizing the low-cost transcriptome sequencing of two parental lines, 'TAC 75' and 'WH 1105', to identify polymorphic SSRs for mapping in a recombinant inbred line (RIL) population. This study introduces a new approach to bridge wheat genetics intricacies and next-generation sequencing potential. It presents a comprehensive genome-wide SSR distribution using IWGSC CS RefSeq v2.1 genome assembly and to identify 189 polymorphic loci through in-silico strategy. Of these, 54.76% showed polymorphism between parents, surpassing the traditional low polymorphic success rate. A RIL population screening validated these markers, demonstrating the fitness of identified markers through chi-square tests. The designed SSRs were also validated for genetic diversity analysis in a subset of 37 Indian wheat genotypes and cross-transferability in the wild/relative wheat species. In diversity analysis, a subset of 38 markers revealed 95 alleles (2.5 allele/locus), indicating substantial genetic variation. Population structure analysis unveiled three distinct groups, supported by phylogenetic and PCoA analyses. Further the polymorphic SSRs were also analyzed for SSR-gene association using gene ontology analysis. By utilizing the developing seed transcriptome data within parental lines, the study has enhanced the polymorphic SSR identification precision and facilitated in the RIL population. The undertaken study pioneers the use of transcriptome sequencing and genetic mapping to overcome challenges posed by the intricate wheat genome. This approach offers a cost-effective, less labour-intensive alternative to conventional methods, providing a platform for advancing wheat breeding research.

Genome sequencing and assembly of Lathyrus sativus - a nutrient-rich hardy legume crop.

Grass pea (Lathyrus sativus) is a cool-season legume crop tolerant to drought, salinity, waterlogging, insects, and other biotic stresses. Despite these beneficial traits, this crop is not cultivated widely due to the accumulation of a neurotoxin - ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in the seeds and its association with neurolathyrism. In this study, we sequenced and assembled the genome of Lathyrus sativus cultivar Pusa-24, an elite Indian cultivar extensively used in breeding programs. The assembled genome of Lathyrus was 3.80 Gb in length, with a scaffold N50 of 421.39 Mb. BUSCO assessment indicated that 98.3% of highly conserved Viridiplantae genes were present in the assembly. A total of 3.17 Gb (83.31%) of repetitive sequences and 50,106 protein-coding genes were identified in the Lathyrus assembly. The Lathyrus genome assembly reported here thus provides a much-needed and robust foundation for various genetic and genomic studies in this vital legume crop.

Genome wide expression profiling of two accession of G. herbaceum L. in response to drought.

BACKGROUND: Genome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of Gossypium herbaceum, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roche's GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions. RESULTS: Fourteen accessions of Gossypium herbaceum were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roche's GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms responses to various abiotic stresses were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death. CONCLUSION: The comparative genome-wide gene expression profiling study of two accessions of G.herbaceum under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.

Development and characterization of genomic and expressed SSRs for levant cotton (Gossypium herbaceum L.).

Four microsatellite-enriched genomic libraries for CA(15), GA(15), AAG(8) and ATG(8) repeats and transcriptome sequences of five cDNA libraries of Gossypium herbaceum were explored to develop simple sequence repeat (SSR) markers. A total of 428 unique clones from repeat enriched genomic libraries were mined for 584 genomic SSRs (gSSRs). In addition, 99,780 unigenes from transcriptome sequencing were explored for 8,900 SSR containing sequences with 12,471 expressed SSRs. The present study adds 1,970 expressed SSRs and 263 gSSRs to the public domain for the use of genetic studies of cotton. When 150 gSSRs and 50 expressed SSRs were tested on a panel of four species of cotton, 68 gSSRs and 12 expressed SSRs revealed polymorphism. These 200 SSRs were further deployed on 15 genotypes of levant cotton for the genetic diversity assessment. This is the first report on the successful use of repeat enriched genomic library and expressed sequence database for microsatellite markers development in G. herbaceum.

Isomaltooligosaccharides utilization and genomic characterization of human infant anti-inflammatory Bifidobacterium longum and Bifidobacterium breve strains.

This study was carried out to understand the probiotic features, ability to utilize non-digestible carbohydrates and comparative genomics of anti-inflammatory Bifidobacterium strains isolated from human infant stool samples. Bacterial strains were isolated from the stool samples using serial dilution on MRS agar plates supplemented with 0.05% l-cysteine hydrochloride and mupirocin. Molecular characterization of the strains was carried out by 16S rRNA gene sequencing. Anti-inflammatory activity was determined using TNF-α and lipopolysaccharide (LPS) induced inflammation in Caco2 cells. Probiotic attributes were determined as per the established protocols. Isomaltooligosaccharides (IMOS) utilization was determined in the broth cultures. Whole genome sequencing and analysis was carried out for three strains. Four obligate anaerobic, Gram positive Bifidobacterium strains were isolated from the infant stool samples. Strains were identified as Bifidobacterium longum Bif10, B. breve Bif11, B. longum Bif12 and B. longum Bif16. The strains were able to prevent inflammation in the Caco2 cells through lowering of IL8 production that was caused by TNF-α and LPS treatment. The strains exhibited desirable probiotic attributes such as acid and bile tolerance, mucin binding, antimicrobial activity, bile salt hydrolase activity, cholesterol lowering ability and could ferment non-digestible carbohydrates such as isomaltooligosaccharides and raffinose. Furthermore, Isomaltooligosaccharides supported the optimum growth of the strains in vitro, which was comparable to that on glucose. Strains could metabolize IMOS through cell associated α-glucosidase activity. Genomic features revealed the presence of genes responsible for the utilization of IMOS and for the probiotic attributes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03141-2.

Evaluating the Distribution of Bacterial Natural Product Biosynthetic Genes across Lake Huron Sediment.

Environmental microorganisms continue to serve as a major source of bioactive natural products (NPs) and as an inspiration for many other scaffolds in the toolbox of modern medicine. Nearly all microbial NP-inspired therapies can be traced to field expeditions to collect samples from the environment. Despite the importance of these expeditions in the search for new drugs, few studies have attempted to document the extent to which NPs or their corresponding production genes are distributed within a given environment. To gain insights into this, the geographic occurrence of NP ketosynthase (KS) and adenylation (A) domains was documented across 53 and 58 surface sediment samples, respectively, covering 59,590 square kilometers of Lake Huron. Overall, no discernible NP geographic distribution patterns were observed for 90,528 NP classes of nonribosomal peptides and polyketides detected in the survey. While each sampling location harbored a similar number of A domain operational biosynthetic units (OBUs), a limited overlap of OBU type was observed, suggesting that at the sequencing depth used in this study, no single location served as a NP "hotspot". These data support the hypothesis that there is ample variation in NP occurrence between sampling sites and suggest that extensive sample collection efforts are required to fully capture the functional chemical diversity of sediment microbial communities on a regional scale.

Metagenomic Sequencing of Multiple Soil Horizons and Sites in Close Vicinity Revealed Novel Secondary Metabolite Diversity.

Discovery of novel antibiotics is crucial for combating rapidly spreading antimicrobial resistance and new infectious diseases. Most of the clinically used antibiotics are natural products-secondary metabolites produced by soil microbes that can be cultured in the lab. Rediscovery of these secondary metabolites during discovery expeditions costs both time and resources. Metagenomics approaches can overcome this challenge by capturing both culturable and unculturable hidden microbial diversity. To be effective, such an approach should address questions like the following. Which sequencing method is better at capturing the microbial diversity and biosynthesis potential? What part of the soil should be sampled? Can patterns and correlations from such big-data explorations guide future novel natural product discovery surveys? Here, we address these questions by a paired amplicon and shotgun metagenomic sequencing survey of samples from soil horizons of multiple forest sites very close to each other. Metagenome mining identified numerous novel biosynthetic gene clusters (BGCs) and enzymatic domain sequences. Hybrid assembly of both long reads and short reads improved the metagenomic assembly and resulted in better BGC annotations. A higher percentage of novel domains was recovered from shotgun metagenome data sets than from amplicon data sets. Overall, in addition to revealing the biosynthetic potential of soil microbes, our results suggest the importance of sampling not only different soils but also their horizons to capture microbial and biosynthetic diversity and highlight the merits of metagenome sequencing methods. IMPORTANCE This study helped uncover the biosynthesis potential of forest soils via exploration of shotgun metagenome and amplicon sequencing methods and showed that both methods are needed to expose the full microbial diversity in soil. Based on our metagenome mining results, we suggest revising the historical strategy of sampling soils from far-flung places, as we found a significant number of novel and diverse BGCs and domains even in different soils that are very close to each other. Furthermore, sampling of different soil horizons can reveal the additional diversity that often remains hidden and is mainly caused by differences in environmental key parameters such as soil pH and nutrient content. This paired metagenomic survey identified diversity patterns and correlations, a step toward developing a rational approach for future natural product discovery surveys.